Operando Raman spectroscopy is a powerful analytical tool to provide new insights in the working and deactivation principles of solid catalysts. Intense fluorescence can obscure Raman spectra to the extent that they become uninterpretable. Time-gated Raman spectroscopy, based on pulsed excitation and time-gated detection, suppresses background fluorescence based on its slower time dynamics compared to Raman scattering. In this work, we demonstrate and quantify the benefit of time gating for operando Raman spectroscopy, using the propane dehydrogenation reaction over Pt–Sn-based catalyst materials as a case study. Experimental time-gated Raman spectroscopy data are fitted to a time-trace model that is used to optimize time gating for the maximum signal-to-background-noise ratio. Time-gated Raman spectra of a spent propane dehydrogenation catalyst material show lower background fluorescence compared to the time-integrated Raman spectra counterparts. Simultaneous operando time-gated and time-integrated Raman spectroscopy experiments demonstrate the benefit of time gating to obtain more distinct Raman features, especially in the early coking stages where spectra are dominated by background fluorescence. Simultaneous time-gated and time-integrated Raman spectroscopy quantifies the benefit of time gating to obtain clearer Raman features in the early coking stages of propane dehydrogenation, when the spectra are dominated by background fluorescence. Operando Raman spectroscopy is a powerful analytical tool to provide new or improved insights in the working and deactivation principles of solid catalysts at work. Spectroscopic information can be interpreted together with online product analysis to couple physicochemical events occurring on the surface of the solid catalyst with its activity, selectivity, and stability.1–3Operando Raman spectroscopy revealed, for example, a correlation between the formation of carbon deposits and catalyst deactivation during e.g., Fischer–Tropsch synthesis (FTS) or propane dehydrogenation (PDH), which are both industrially relevant catalytic processes.4–67 At some point, the signal-to-noise ratio (SNR) of Raman bands can become so low that the bands are indistinguishable from the background, resulting in spectra that are uninterpretable.4,5Raman scattering is an intrinsically weak process, so that its spectral features are sometimes overshadowed by other signals reaching the detector. Intense background signals may arise from sample fluorescence, ambient light, or black body radiation. In practice, Raman spectra processing often includes a background subtraction. This can, however, be challenging if the background signal has a particular spectral shape. Moreover, random noise cannot be subtracted and becomes higher with increasing background intensity.operando spectroscopy purposes.8–11 The background fluorescence can also be reduced by shifting to longer laser excitation wavelengths. This makes electronic transitions in organic molecules, resulting in fluorescence, less likely. Shifting to lower excitation energy however lowers the chance of a Raman scattering event.12 Alternatively, UV lasers are used as excitation sources to avoid background fluorescence. In this case, background fluorescence induced by the excitation of aromatic molecules appears at longer wavelengths than the Raman features.13 This principle has been applied for the study of coke formation during the methanol-to-olefins (MTO) reaction.14,15Several techniques have been developed to improve the quality of Raman spectra. Common strategies involve the enhancement of Raman signal. For instance, nanostructured plasmonic surfaces or plasmonic nanoparticles can boost the Raman signal intensity, but the stability of such nanostructures is still limited, especially at elevated temperatures, thereby hampering their applicability for 16 By time gating the detection, background fluorescence and Poisson noise on it are reduced and the spectral quality is improved. Fluorescence can be rejected using an optical Kerr gate17,18 or using detectors with a high time resolution, typically complementary metal-oxide-semiconductor single-photon avalanche diodes (CMOS-SPAD).12,19–21Time-gated Raman spectroscopy offers improved spectral quality over conventional Raman spectroscopy. It suppresses background fluorescence based on its slower time dynamics compared to Raman scattering. Raman scattering occurs instantaneously after excitation, whereas fluorescence of organic molecules typically exhibits a lifetime on the ns scale.22 and carbon deposits on other strongly fluorescent zeolite-based catalyst materials,23,24 as well as in battery research.25,26 CMOS-SPAD-based time-gated Raman spectroscopy was used for numerous different type of samples, including biological samples and pharmaceuticals,27–31 rare-earth-element containing rock samples,32 high-temperature melts,33 and pickle liquor.34Time-gated Raman spectroscopy is applied in a wide variety of fields yielding insightful results. Kerr-gated Raman spectroscopy (KG-RS) has been applied for the study of carbonaceous species on solid catalysts, including the formation of coke species during the MTO reaction27,28,32 A few studies quantitatively assed the effect of time gating based on CMOS-SPAD detectors.35,36 Kekkonen et al. reported a 4.4–8.8 fold higher signal-to-peak-to-peak-noise ratio (SNRpp) in time-gated compared to conventional Raman spectroscopy on a tooth sample. Kotula et al. used the ratio of background-corrected Raman signal to the standard deviation in the background (SBNR) as a quality descriptor and reported an 8–68-fold increase in spectral quality from recovered plastics. However, switching between two spectroscopy modes inevitably results in working with two spectrometers with different designs and different optical paths. A quality descriptor that depends on photon-count rates, such as the SNR, tests not only the effect of time gating but also the detection efficiency of the experimental setup.35,36Precisely assessing the beneficial effect of time gating on spectral quality remains challenging. In some studies, time-gated Raman spectroscopy was compared with conventional Raman spectroscopy using a qualitative approach.operando Raman spectroscopy for studying solid catalysts at work. We follow a Raman-microscopy-based approach for the ex situ analysis of spent PDH catalysts. Based on an analysis of the time dynamics of Raman scattering and background fluorescence upon pulsed laser excitation, we select the time gate for an optimal signal-to-background-noise ratio (SBNR). Optimally time-gated Raman spectra are compared to time-integrated Raman spectra recorded with a pulsed laser and a CCD detector. The lessons learned are applied in an operando simultaneous time-gated and time-integrated Raman spectroscopy experiment of a PDH catalyst material in a laboratory-scale reactor. The results illustrate the urgency to consider sample heterogeneity for analyses of the catalyst materials under study and highlight that time gating lowers the background fluorescence, to make Raman features better distinguishable.In this work, we demonstrate and quantify the benefit of CMOS-SPAD-based time gating for 16 Pulsed excitation is used to discriminate between the two events based on their differences in time dynamics. Both signals are convoluted with the instrument response function (IRF), which we approximate as a Gaussian. The resulting time traces are shown in 35,36 In this work, we have selected two quality descriptors. For the signal-to-background-noise ratio (SBNR), the signal is defined as the amplitude of a Raman band compared to the standard deviation on the background signal, calculated using the noise characteristics of the detectors. The model used in this section assumes Poisson noise, so that the background noise equals the square root of the background counts. The SBNR can be regarded a quantitative descriptor for the spectrum that illustrates the Poisson noise suppression due to time gating. A drawback of this parameter is that it is not intrinsic, but depends on the integration time and the efficiency of the detector system. In addition, we have opted for the Raman-to-total ratio (RTR): the ratio of the amplitude of the Raman band above the background to the total signal in the Raman band. This parameter describes the shape of the spectrum and is independent of the number of counts.Choosing the proper quality descriptor for spectral quality is key for drawing conclusions about the effectiveness of time gating.1919,42 Time gating reduces the background by 96% and the background noise by 81%, while the Raman signal decreases by 27%. This improves the SBNR of the time-gated spectrum to 9.4, compared to 2.5 for the time-integrated spectrum.The time-trace model can predict which time gating settings yield the optimal spectrum.operando spectroscopy experiments. A typical time-resolved Raman spectrum recorded on spent and coked catalyst material is shown in t = 0 ns is highest at 1320 and 1600 cm−1. These peaks, associated with coke deposits, originate from the Raman D-band associated with ring breathing modes of the edges and defects of graphitic sheets and the Raman G-band due to ring breathing modes of graphitic sheets, respectively.5 The ratio between de bands as well as the exact position and width can be used to deduce the chemical nature of the carbon deposits.5 We further observe spectrally broad background signal, which decreases fast (sub ns) and has a low-intensity tail that decays on the timescale of a few ns. This is assigned to fluorescence of carbonaceous species on the surface of the spent catalyst material.−1, i.e., the area of the G-band. To determine the dynamics of solely Raman scattering, we use a reference measurement on a crystalline silicon wafer  and is therefore preferable for many applications, such as an operando spectroscopy investigation of solid catalysts. t1 and t2, yield the optimal Raman spectrum. We define the SBNR as:where S is the Raman signal in the G-band obtained by integrating the Raman intensity IR. σB is the Poisson noise on the fluorescent background intensity IF. The counts are presented after a sensitivity correction, that effectively multiplied the counts in the studied region of the spectrum with 1.5. The calculated noise is corrected for this modulation with the correction factor CF  and G (1600 cm−1) bands, associated with coke deposits, are clearly visible in both heatmaps. In addition, a band at 1050 cm−1 is observed that can be ascribed to Si–O vibrations of the quartz reactor.5 The time-gated data show some broadband background fluorescence between 1800 and 2000 cm−1 at the beginning of the experiment, which disappears within 30 min. The initial intense fluorescence is more pronounced in the time-integrated data. Here, the D and G band are barely distinguishable above the background fluorescence in the first 30 min of the experiment. The initial fluorescence is likely caused by polycyclic aromatic molecules that are the precursors of coke deposits. In the initial stages of the reaction, aromatics are formed by dimerization of propyl species due to side reactions, such as cracking and deep dehydrogenation.43,44 In later stages, the aromatics grow into graphitic sheets covering the catalyst and losing their fluorescence.43,44 Indeed, the heatmaps of non-normalized spectra reveal that the absolute fluorescence intensity peaks after 2 min after which it decreases with time reaching half its intensity after 48 min. For the Pt-based catalyst the fluorescence decreases instantly and halves after 2 min , 0.5 wt% Pt 1.5 wt% Sn/Al2O3 , Pt(NO3)2·nH2O , Sn(ii)Cl2·2H2O , Rhodamine 6G  and γ-Al2O3 .The following solid catalysts and other materials have been used in this study: 0.5 wt% Pt/Al2O3 catalyst was prepared by impregnating an aqueous Pt(NO3)2 solution onto the Al2O3 support. The impregnated catalyst was subsequently dried at 120 °C for 30 min and calcined in air at 560 °C for 3 h. The 0.5 wt% Pt 1.5 wt% Sn/Al2O3 was prepared by impregnating an aqueous Pt(NO3)2 solution onto the Al2O3 support. The impregnated catalyst was subsequently dried at 120 °C for 30 min. In a second impregnation step the Sn(ii)Cl2 was impregnated onto the catalyst, dried at 120 °C for 30 min and calcined in air at 560 °C for 3 h.The 0.5 wt% Pt/Alet al.  to reduce the catalyst to its catalytically active metallic state. The reactor was then purged with He (8 mL min−1) for 5 min. The dehydrogenation started when propane was introduced to the reactor at a flow rate of 8 mL min−1. At the same time, the online gas chromatography (GC) program as well as the simultaneous time-resolved time-integrated and time-gated Raman spectroscopy experiments (see below) were initiated. A gas chromatogram (GC) was recorded every 6 min with a Compact GC 4.0  equipped with two Rt-UBond columns (2 m × 0.32 mm and 8 m × 0.32 mm) and a flame ionization detector (FID). After 4 h of reaction, the gas feed was switched to He (8 mL min−1) and the oven was cooled down to room temperature. The samples were collected for ex situ analysis.Propane dehydrogenation (PDH) experiments were performed in a lab-scale reactor with a method adapted from Sattler . Fig. S1.5,37 300The Raman spectroscopy experiments presented in this work were carried out with two types of spectrometers. The time-integrated spectra were recorded with the AvaRaman-532 HERO-EVO (Avantes) spectrometer equipped with a 25 μm slit, a HSC1200-0.75 grating, and a back-thinned TE cooled CCD Detector with 1024 × 58 pixels. The time-gated spectra were recorded with the PicoRaman M1 (Timegate Instruments) equipped with 768 × 8 SPAD pixels. The pulsed 532 nm laser  of the PicoRaman was used as the excitation source. We refer to the devices as time-integrated and time-gated spectrometers.i.e., the recorded spectrum as a function of delay time after the laser pulse. The detector can be activated up to about 6 ns after the arrival of the laser pulse. The software scans through the range of delay times multiple times to avoid artefacts due to sample degradation occurring on the time scale of minutes. We report the number of laser pulses used to probe the Raman spectrum at each delay time.In the case of time-gated Raman spectroscopy, a pulsed laser is used to excite the catalyst sample, which typically induces both Raman scattering and fluorescence. The resulting signal is guided towards the time-gated spectrometer with optical patch cables. After entering the time-gated spectrometer, the signal pulse falls on a dispersion element. Light with wavelengths between 515 and 600 nm falls on the detector composed of 8 × 768 SPAD pixels (6 sub detectors with 8 × 128 pixels each). The time gating occurs at the detector. The SPAD pixels are switched on after a delay time with respect to the laser pulse. By doing so, a fraction of the signal pulse is recorded. All 768 pixels in the wavelength dimension are equipped with 8 sub pixels, constituting 4 pairs that are sequentially activated for 100, 200, 100, and 1000 ps, respectively. By combining the signal of the different detector pairs, one has access to time gates of 100, 200, 300, 400, 1000, 1100, 1300, and 1400 ps. A time-gated Raman spectrum is obtained by counting the photons that arrive within the time bins after the set delay time, for a set number of laser pulses. This process is repeated for a range of delay times yielding a time-resolved Raman spectrum, via the same optical path. A 50× 0.8 NA Olympus objective with a working distance of 0.66 mm was used for the measurements. The pulsed laser of the PicoRaman was used as the excitation source, delivering a power of 6.2 mW (measured with an Ophir Orion PD power meter) and illuminating an area of 41 μm in diameter.A spent Pt–Sn-based PDH catalyst material was analyzed with time-gated and time-integrated Raman microscopy Fig. S1. An upri2O3 PDH catalyst. First, a time-gated Raman dataset was recorded with the full range of delay times to obtain as much as possible of the temporal shape of the signal pulse. The time-gated spectra were recorded for delay times between 4.30 and 10.21 ns in 396 steps of 15 ps. At each delay time, the spectrum was integrated for 70 000 laser pulses at a repetition rate of 150 kHz resulting in a total acquisition time of 185 s. Subsequently, the collection path was switched to the time-integrated spectrometer to record a time-integrated spectrum, while using the same excitation source and power. Here, we used an integration time of 1.87 s, which is shorter than the total acquisition time of the time-resolved Raman spectrum by a factor 396/4. By doing so, the acquisition time of the time-integrated spectrum is equal to that of the sum of time-gated spectra of four delay times. The time-integrated spectrum and the sum of four time-gated spectra can be directly compared as the acquisition time, location on the sample, laser spot size, and laser power used were equal. The recording of the spectra is preceded by the acquisition of a spectrum with the same settings in the absence of the excitation source. This measurement is denoted as the dark spectrum. Reflection images of the spot before and after laser exposure were recorded with a CS165CU/M 1.6 MP Color CMOS Camera (Zelux) that was integrated in the Microprobe. The images were used to assess laser-induced sample degradation. The procedure was followed to analyze 15 spots.The microscopy set-up was used in a procedure developed to study the temporal shape of the Raman signal and directly compare time-gated and time-integrated Raman spectroscopy. The sample under study was the coked 0.5 wt% Pt 1.5 wt% Sn/Aloperando time-gated and time-integrated Raman spectroscopy. We subjected the Pt- and Pt–Sn-based catalyst materials to the PDH procedure described above. The quartz reactor was equipped with a rectangular window that was aligned with a hole in the oven that allowed for the study of the catalyst bed with a fiber-coupled optical probe. An optical patch cable was used to deliver the light from the time-gated spectrometer to the probe . The probe was composed of a HFPH-FC-S-532 filter box  and an in-house designed head based on a 9.525 mm diameter sapphire ball lens (Edmund Optics Inc.) that focused the laser light on the sample at 0.71 mm and collected the resulting signal. The laser power at the probe head was 25 mW, measured with an Ophir Orion PD power meter. The collected signal passed through the head, the filter box, and an optical patch cable  and was split with a 1 × 2 optical patch coupler  to deliver the signal to the time-gated and time-integrated spectrometer simultaneously.The formation of carbon deposits on PDH catalyst material was studied with simultaneous 2 and heated up to 600 °C, after which it resided in a He flow. During this step, both types of Raman spectra were recorded in the absence of laser light to be used for the background correction. Hereafter, propane was introduced to the system for 4 h. At the same time, the online GC program as well as the simultaneous time-resolved time-integrated and time-gated Raman experiments were initiated. Half of the signal was delivered to the time-integrated spectrometer where a spectrum was recorded every minute . The other half to the signal was recorded with time-gated spectrometer. The time-gated spectra were recorded for 5 delay times; 5.37, 5.38, 5.39, 5.40, and 5.41 ns. At each delay time, the spectrum was integrated for 884 975 laser pulses at a repetition rate of 150 kHz resulting in a total experiment time of ∼30 s. Effectively, however, we recorded one spectrum every minute when using these settings, due to software related delay during the initiation of a measurement and the processing of the results.The catalyst was activated in H38–40 A close inspection showed that, after delay correction, the time-gated Raman spectra exhibited Poisson noise  to compensate for non-uniformity in pixel response of the SPAD array. The ‘delay correction’ corrects for width and offset variations in the time bins. Convolution compensates for both while translation only compensates for the offset variation. In the literature, pixel-to-pixel timing heterogeneity, so called timing skew, is estimated to cause more distortion in the time-gated spectra than Poisson noise.e Fig. S2. Furtheroperando and microscopy experiments. The recorded wavelengths (nm) were converted to Raman shift (cm−1) using the laser wavelength of 532.21 nm  was used to acquire spatial information on the nature of the carbon deposits on the surface of the spent Pt–Sn-based catalyst material. A 50× 0.5 NA objective with a working distance of 10.6 mm was used to focus a 532 nm laser that delivered 0.52 mW to the sample (measured with an Ophir Orion PD power meter) illuminating a diffraction-limited spot. A long pass edge filter in the detection path was used to block the laser light and a 1200 lines per mm grating was used as dispersion element.et al.41 The light of a deuterium/halogen lamp (AvaLight-DH-S-BAL) was led towards the spectrometers under study via optical patch cables and a cube that facilitated the placement of several neutral-density filters in the optical path. Subsequently, we have recorded a large number of spectra for several illumination intensities. The time-integrated spectrometer was used to record 2500 spectra with 100 ms integration time. The time-gated spectrometer was used to record 250 spectra. The time-gated spectra were recorded for 5 delay times; 4.66, 4.67, 4.68, 4.69 and 4.70 ns. At each delay time, the spectrum was integrated for 350 000 laser pulses at a repetition rate of 150 kHz.The photon counting noise of both spectrometers was characterized with a method adopted from van Swieten We have explored, optimized and validated time-gated Raman spectroscopy based on the signal dynamics of the G-band due to graphitic carbon deposits on spent propane dehydrogenation catalyst materials. We were able to distinguish the G-band Raman scattering from background fluorescence based on their different time dynamics. A time-trace model helped us determine the time gating that yields the optimal signal to background noise ratio. A fast, sub ns component of the fluorescence made complete suppression of background fluorescence by time gating difficult.Optimally time-gated Raman spectra of a spent propane dehydrogenation catalyst showed clearly lower background fluorescence compared to the time-integrated Raman spectra. As the two detectors exhibited different noise characteristics, the effect of time gating could be best assessed in terms of the Raman-to-total ratio, instead of the signal-to-background-noise ratio. The spot-to-spot variations showed that the effect of time gating is location-dependent and stresses the importance of measuring on the exact same spot when comparing the two detection techniques.operando time-gated and time-integrated Raman spectroscopy experiments demonstrated the benefit of time gating. The Raman-to-total ratio of the spectra recorded with time gating is higher than the time-integrated spectra, especially in the beginning of the propane dehydrogenation reaction over Pt/Al2O3 and Pt–Sn/Al2O3 catalysts. These results highlight that measuring in a time-gated fashion enables better distinction of the Raman features, especially in the early coking stages of solid catalysts where Raman spectra were dominated by background fluorescence.The simultaneous 45,46 The results obtained are shown in Fig. S9.To illustrate the versatility of the analytical approach proposed, we have explored the potential of time-gated Raman spectroscopy for zeolites. Classical Raman spectroscopy of zeolite-based materials is known to be complicated by a broad and intense background fluorescence.B. M. W. conceived the initial ideas of the PhD project, wrote the project, and acquired the related funding. B. M. W. and F. T. R. designed and supervised the research. R. V. performed the experimental work and analyzed the data. R. V., P. T. P. and F. T. R. constructed the time-trace model. R. V., P. T. P., F. T. R. and B. M. W. contributed to the interpretation of the results. R. V. wrote the manuscript with feedback from all authors.The authors declare no conflict of interest.CY-013-D3CY00967J-s001


PD-L1 analysis in TNBC is essential for selecting patients eligible for immunotherapy. Limited data are available on pathologists’ concordance regarding PD-L1 assessment. Twelve pathologists of various expertise from three European countries digitally analysed 100 breast cancer core biopsies stained using the SP142 PD-L1 assay in two rounds. The overall inter-observer agreement among the pathologists was substantial. The intra-observer agreement was substantial to almost perfect. The expert scorers were more concordant in evaluating staining percentage compared with those of the non-experts. Challenging cases around the 1% cut-off value for positivity were identified and represented a small 6–8%) proportion of all cases. The experts were more concordant in scoring those cases. The study shows reassuringly strong inter- and intra-observer concordance among pathologists in PD-L1 scoring. A proportion of low-expressors remain challenging to assess, and these would benefit from addressing the technical issues, testing a different sample and/or referring for expert opinions.2 = 0.920 vs. 0.890). Discordance predominantly occurred in low-expressing cases around the 1% value. Some technical reasons contributed to the discordance. The study shows reassuringly strong inter- and intra-observer concordance among pathologists in PD-L1 scoring. A proportion of low-expressors remain challenging to assess, and these would benefit from addressing the technical issues, testing a different sample and/or referring for expert opinions.The assessment of PD-L1 expression in TNBC is a prerequisite for selecting patients for immunotherapy. The accurate assessment of PD-L1 is pivotal, but the data suggest poor reproducibility. A total of 100 core biopsies were stained using the VENTANA Roche SP142 assay, scanned and scored by 12 pathologists. Absolute agreement, consensus scoring, Cohen’s Kappa and intraclass correlation coefficient (ICC) were assessed. A second scoring round after a washout period to assess intra-observer agreement was carried out. Absolute agreement occurred in 52% and 60% of cases in the first and second round, respectively. Overall agreement was substantial (Kappa 0.654–0.655) and higher for expert pathologists, particularly on scoring TNBC (6.00 vs. 0.568 in the second round). The intra-observer agreement was substantial to almost perfect (Kappa: 0.667–0.956), regardless of PD-L1 scoring experience. The expert scorers were more concordant in evaluating staining percentage compared with the non-experienced scorers (R Advances in biomarker assessment, companion diagnostics and genomics have revolutionised the way breast cancer is currently classified and managed ,2. The iThe limited data available in the literature on non-breast cancer suggest the poor reproducibility of PD-L1 SP142 scoring . Some stWe therefore aimed to assess the inter- and intra-observer concordance of breast pathologists of various expertise and geographical locations in reporting a large cohort of PD-L1 SP142-stained invasive breast carcinomas of various molecular subtypes to assess if particular molecular subtypes would be more or less prone to poor inter-observer concordance. We also sought to analyse discordant cases in detail to gain insight into the reasons for discrepancies in PD-L1 results, allowing for a subsequent search for strategies on how to tackle them.Core biopsies from a total of 100 cases of primary breast cancers were included in the study. Cases were selected retrospectively from the files of a single large UK institution  to include all molecular subtypes with enrichment for the TNBC group.https://eslidepath.bham.ac.uk, Last accessed 23 February 2023. Each participant was provided with a unique username and password to allow for access to the digital platform for whole slide scoring. Twelve pathologists from eight institutions representing three European countries  evaluated all cases in round one, of whom 10 re-scored the same cases in round two, separated by at least 3 months of a washout period designed to assess intra-observer variability. All pathologists had previously received Roche training for SP142 PD-L1 scoring in TNBC and passed a proficiency test.First, 4 μm sections of formalin-fixed, paraffin-embedded tumour blocks were cut and stained using the VENTANA SP142 anti-PD-L1 rabbit monoclonal primary antibody and a VENTANA Benchmark ULTRA automated staining platform, according to the manufacturer’s protocol. A section from a cell block containing three cell lines with various staining intensities and a section of normal tonsil were included as on-slide controls. Paired H&E sections and PD-L1-stained immunohistochemistry slides were digitally scanned using a Leica Aperio AT2 slide scanner at x40 and uploaded to the University of Birmingham digital platform via a secure link: PD-L1 SP142 Immune cell (IC) scoring was conducted according to the recommended scoring algorithm , using aThe data were tabulated and statistically analysed using the SPSS (IBMS) software version 28. We used standard statistical analyses for assessing intra/inter-rater concordance/agreement, which have been previously described . IntraclFleiss multiple-rater Kappa statistics of inter-observer and intra-observer agreement for designating cases as PD-L1-positive versus -negative using a cut-off value of 1% were calculated. Fleiss’ Kappa κ is a measure of inter-rater agreement used to determine the level of agreement between two or more raters when the method of assessment, known as the response variable, is measured on a categorical scale. The Kappa results are interpreted as follows: values ≤ 0 indicate no agreement, values from 0.01 to 0.20 indicate none to slight agreement, values from 0.21 to 0.40 indicate fair agreement, values from 0.41 to 0.60 indicate moderate agreement, values from 0.61 to 0.80 indicate substantial agreement and values from 0.81 to 1.00 indicate almost perfect agreement.A case was regarded as PD-L1 positive or -negative if more than 50% of the participants designated it as positive or negative, respectively. The consensus score was considered a majority score if 67% or more of the participants agreed on the categorisation. If all participants agreed (100%), this was regarded as absolute agreement (AA). The cases with agreement less than 67% and above 50% were considered challenging. In cases of no agreement (50% or less), a case was considered as PD-L1-positive or -negative based on the consensus of the experienced pathologists only.Scatter plots were used to visualise percentage PD-L1 scores, and the strength of the relationship between scores was expressed as a squared correlation coefficient (R2). All analyses were supervised by an expert in pathology informatics (PL).An outline of the study methodology is shown in A total of 100 breast cancers were assessed, comprising 29/93 (33.3%) grade 2 and 62/93 (66%) grade 3 cases, while 7 cases had missing grades. The patient ages ranged from 42 to 59 years, with a median of 49 years. Fifty-eight carcinomas were triple-negative, 28 were luminal and 14 were Her2-positive. All cases were evaluated independently by twelve pathologists, including nine specialist consultant breast pathologists, of whom six had 1–3 years’ experience in PD-L1 scoring, as per the survey responses. All scorers had significant experience in breast pathology reporting, and six had experience in scoring PD-L1 SP142 in TNBC in routine practice. A consultant Biomedical Scientist and two trainee pathologists were among the scorers. Ten pathologists scored round two, following a washout period. The overall percentage of PD-L1 positivity for all of the breast cancer molecular subtypes was 36–38%, and the highest was for TNBC (55%) .The Kappa of the inter-observer agreement between the participants in classifying cases as PD-L1-positive vs. -negative and the number of cases with absolute agreement (AA) for rounds one and two were calculated. The overall agreement was substantial  .There was absolute agreement on scoring cases as either positive or negative in 52 cases in the first round A–D. This2 = 0.920 vs. 0.89). The overall concordance was excellent (R2 = 0.935). The distribution of the percentage scoring among all raters (those with and without experience in PD-L1 routine reporting) in both rounds is shown in When the median percentage of PD-L1 expression was considered, the expert pathologists had a higher and tighter concordance compared with the non-experts  or no agreement (<50%), most of which (8/10) were of the TNBC phenotype . All tenIt is of note that the consensus in scoring those challenging cases among the expert pathologists ranged from no agreement (50%) to absolute agreement (100%), with a higher percentage of the former in the first round  compared to the second . For the experts, concordance improved in the second round, with all but one case showing absolute agreement. Strong to almost perfect agreement among the experts was seen in 6/10 of those challenging cases, in both rounds. For the non-experts, the proportion of cases with low or no agreement was higher than that for the experts and increased from 30% in the first round to 50% in the second round .Cohen’s Kappa was calculated to assess the inter-observer agreement between each of the scoring pathologists and the intra-observer agreement for each scorer across the two rounds. Inter-observer agreement was, overall, moderate (0.5) to substantial (0.75) . The higICC was used to assess the reliability of scoring between different groups of raters using the median percentage expression. The ICC for different groups  ranged from moderate (0.5–0.75) to excellent (>0.9), with the predominance of the latter . The higAll scorers had significant experience in breast pathology reporting, but only six scored PD-L1 SP142 in breast cancer in routine practice. The experience in PD-L1 reporting did not appear to affect the intra-observer agreement, with all scorers showing substantial or almost perfect agreement. On the other hand, the intra-observer reliability in the percentage assessment of PD-L1 expression was higher for experienced pathologists compared with non-experienced pathologists .We present comprehensive data of a large PD-L1 concordance cohort, scored twice by pathologists from eight institutions, representing three countries. Our data show reassuring inter- and intra-observer agreements, which were the highest among experts, and highlight cancers with low levels of PD-L1 expression as the most challenging in classifying as either PD-L1-positive or -negative.Unlike standard diagnostic and prognostic markers for breast cancer, SP142 PD-L1 immunohistochemistry is assessed in the immune micro-environment of breast cancer and not in the neoplastic cells themselves. PD-L1 expression in foci of ductal carcinoma in situ (DCIS), necrotic debris, normal mammary tissue and normal nodal tissue is excluded. Therefore, experience in both tumour morphology and PD-L1 assessment is required and may affect the reproducibility of scoring.Few studies, summarised in While several antibodies/assays for PD-L1 assessment are available , the VENTANA Roche SP142 assay is the only companion FDA- and CE-IVD (European Commission in vitro diagnostics)-approved test for atezolizumab therapy. An expert round table in 2019  recommenIn this study, we assessed both the inter- and intra-observer concordance among the participating pathologists. It is notable that the intra-observer concordance was high (0.667 to 0.956) among both expert and non-expert pathologists in PD-L1 scoring, indicating that pathologists are likely to stick to their parameters on scoring. When the median percentage of PD-1 expression was compared among the raters, the highest ICC (0.974) was achieved among experienced raters in the second round. We observed the lowest concordance value of 0.619 when comparing non-experienced to experienced scorers. Similarly, a higher concordance among those experienced in PD-L1 scoring (93.3%) compared with non-experts (81.5%) was previously reported by Pang et al. .While, overall, there was a high concordance among pathologists in PD-L1 SP142 scoring, some cases were challenging to score. Those cases comprised 6–8% of all cases and generally showed very low levels of expression spanning the threshold for positivity. These may represent a so called “borderline category” where expression cannot readily be designated into a clear-cut positive or negative status. Ideally, information on the tumour response to immunotherapy should determine how those cases should be classified. It is of interest that expert pathologists, who routinely reported PD-L1 in breast cancer, showed substantial concordance in scoring those difficult cases. We therefore recommend that those cases of very low expression  are scored by an expert pathologist either via double-reporting or via a second opinion referral.Similar challenges in PD-1 scoring have been highlighted in carcinomas in other tissues. For example, the concordance between the assays used for PD-L1 assessment in head and neck squamous cell carcinoma (HNSCC) was fair to moderate, with a tendency for the SP142 assay to better stain the immune cells . FurtherOur data also confirm previous studies showing the highest proportion of PD-L1 positivity in TNBC . PD-L1 wThe current study and previous lessons from the IMpassion trial  shed somIn summary, we present a detailed analysis of 12 pathologists who scored 100 digitally scanned breast cancer slides for PD-L using the Ventana SP142 assay in two rounds separated by a washout period. Absolute (100%) agreement was substantial in 52% and 60% of cases in the first and second rounds, with Kappa values of 0.654 and 0.655 for rounds one and two, respectively. We provide reassuring evidence of a high concordance of PD-L1 reporting among pathologists, the highest being among experts and in reporting challenging, low-expressing TNBC. The intra-observer agreement was substantial for all raters. Despite experience and the adherence to current reporting guidelines, there remains a minority of tumours (6–8%) that are challenging to assign to either a positive or negative category. Those are PD-L1 low-expressing and/or heterogeneous tumours that suffer from the least concordance among pathologists. Consensus scoring and referrals for expert opinions should be considered in those cases. If uncertainly persists, this should be recognised and well communicated to clinicians in the context of a multidisciplinary approach. For inconclusive cases, testing on another tumour sample and/or using another assay  could be performed.Pathologists’ training and experience are paramount in evaluating PD-L1 expression and selecting patients for immune checkpoint anti-PD-L1 inhibitors. Further work on refining the criteria for scoring, pathologists’ training and assessing pathologist concordance is needed. This will ensure the accurate classification of tumours into a positive or negative category and, hence, the accurate selection of patients for atezolizumab therapy.This study also shows that digital pathology is a useful tool that allows for the instantaneous sharing of high-quality whole slide scans with colleagues. This is particularly helpful for consensus scoring and/or seeking expert opinions.


To compare the inter-modality consistency and diagnostic performances of the contrast-enhanced ultrasound (CEUS) Liver Imaging Reporting and Data System (LI-RADS) and contrast-enhanced computed tomography (CECT) LI-RADS in patients at risk for hepatocellular carcinoma (HCC), so as to help clinicians to select a more appropriate modality to follow the focal liver lesions (FLLs).This retrospective study included untreated 277 FLLs from 247 patients who underwent both CEUS and CECT within 1 month. The ultrasound contrast medium used was SonoVue. FLL categories were independently assigned by two ultrasound physicians and two radiologists using CEUS LI-RADS v2017 and CECT LI-RADS v2018, respectively. The diagnostic performances of CEUS and CECT LI-RADS were evaluated using sensitivity, specificity, positive predictive value (PPV), and negative predictive value. Cohen’s Kappa was employed to evaluate the concordance of the LI-RADS category.p < 0.001). HCC was more frequently observed in CECT LR-3 and LR-4 hepatic lesions than in CEUS . The specificity and PPV of CEUS and CECT LR-5 for the diagnosis of HCC were 89.5%, 95.0%, and 82.5%, 94.4%, respectively. The sensitivity of CEUS LR-5 + LR-M for the diagnosis of hepatic malignancies was higher than that of CECT . The specificity and PPV of CEUS LR-M for the diagnosis of non-HCC malignancies were lower than those of CECT .The inter-modality consistency for CEUS and CECT LI-RADS was 0.31 (The inter-modality consistency between the CEUS and CECT LI-RADS categories is fair. CEUS LI-RADS was more sensitive than CECT LI-RADS in terms of identifying hepatic malignancies, but weaker in terms of separating HCC from non-HCC malignancies.The online version contains supplementary material available at 10.1186/s12880-023-01088-1. The early identification of hepatocellular carcinoma (HCC) is essential for improving patient prognosis . ContrasBoth CEUS LI-RADS and CT/MRI LI-RADS are used to categorize FLLs in patients who are at a high risk of developing HCC. Even so, it is unknown whether the use of LI-RADS in the interpretation and reporting of FLLs differs depending on which of these two different imaging modalities is used. It is therefore important to compare the inter-modality consistency of LI-RADS from CEUS and CECT as different LI-RADS categories may significantly affect treatments and follow-up plans. Although there have been some previous reports on the concordance of CEUS LI-RADS and CT/MRI LI-RADS, none have compared CEUS LI-RADS with CECT LI-RADS alone to our knowledge –9. It isThis study was approved by the Ethics Committee of our hospital; the requirement for individual consent was waived. Patients in our hospital who had undergone both CEUS and CECT liver examinations and had a histopathology of the FLLs from November 2020 to October 2021 were selected (n = 510). A total of 263 patients were excluded for the following reasons: an interval of more than one month between the CEUS and CECT (n = 11), prior treatment for FLLs (n = 82), having FLLs that could not be shown on either US or CECT (n = 9), not being at high risk for HCC as defined by ACR LI-RADS (n = 144), an unclear pathological diagnosis of FLLs (n = 12), and poor image quality (n = 5). Ultimately, 277 FLLs from 247 patients  were included in this study.The conventional ultrasound and CEUS examination of the patients’ FLLs was performed using the Mindray RESONA7  and GE LogiqE9  ultrasound diagnostic instruments. The convex array probe used a frequency range of 1–6 MHz. Harmonic imaging and low mechanical index (MI 0.07–0.10) were applied for CEUS of the liver. The contrast agent  was prepared in 5 ml of 0.9% saline and shaken well. The conventional contrast dose used for the liver CEUS was 1.5 ml. A scan of the whole liver was performed prior to starting the CEUS, and any FLLs found were assessed for size, borders, echogenicity, and blood flow, among other factors. Timing started after the antecubital vein bolus of the contrast agent; we recorded dynamic images of CEUS for the first 60 s, and then stored static images with typical enhanced features at 20–30 s intervals. The CEUS examination was dynamically observed for either 3–5 min or until the microbubbles disappeared. If multiple nodules were encountered, repeat contrast injections were required to visualize the remaining FLLs. Each patient’s CEUS images were then exported to mobile hard drives for subsequent analysis.A dual-source CT  was used to perform both a nonenhanced scan of the patient’s FLLs and scans of the arterial, portal, and late phases. The scanning parameters were as follows: 120 kVp, 400 mAs, layer thickness of 1.5 mm, layer spacing of 1.0 mm. A syringe pump was used to inject the contrast agent  intravenously at a dose of 1.5-2.0 ml/kg of the patient’s body weight and a rate of 3.0-3.5 ml/s. Images of the arterial, portal, and late phases were acquired at 25–30 s, 60–70 s, and 120 s, respectively, after the contrast injection. The CECT images were then stored in the PACS for subsequent analysis.https://www.acr.org/Clinical-Resources/Reporting-and-Data-Systems/LI-RADS). The four physicians assigned the LI-RADS categories independently; although some of the patients’ images had been interpreted by the physicians at the time of the patient’s original visit, the time between the last image interpretation and the current image interpretation was long. Moreover, all of the physicians were unaware of the patients’ clinical information and pathological findings at the time of image interpretation. In clinical practice, if different LI-RADS classifications were obtained for the same FLL in two imaging methods, patients were generally managed or followed up with the higher LI-RADS classification. In order to minimize the effect of inexperience on interpretation, the results of the senior physicians’ interpretations were used to choose between the two imaging methods (Reader 1 vs. Reader 3). We also assessed the concordance of the LI-RADS category between the differently experienced physicians (Reader 1 vs. Reader 2 and Reader 3 vs. Reader 4).The CEUS images were interpreted by two ultrasound physicians with different levels of experience; Reader 1, W.Y.Q., had over 10 years of liver imaging experience while Reader 2, W.R., had over 3 years of experience at the time of interpretation. The CECT images were interpreted by two radiologists with different levels of experience; Reader 3, S.R., had over 15 years of abdominal CT imaging interpretation experience while Reader 4, Q.L.H., had over 3 years of experience at the time of interpretation. The imaging physicians reviewed the CEUS LI-RADS v2017 and CT/MRI LI-RADS v2018 guidelines prior to interpreting the contrast images. The CEUS and CECT diagnostic tables for LR-3, LR-4, and LR-5 are shown in Fig. p < 0.05.Quantitative data was expressed as the mean ± standard deviation while ranges and qualitative data were expressed as frequencies and percentages. Kappa (κ) and a 95% confidence interval (CI) were used to assess the consistency of the LI-RADS category. Different levels of concordance corresponded to different κ values as follows: 0 to 0.20 - poor concordance; 0.21 to 0.40 - fair concordance; 0.41 to 0.60 - moderate concordance; 0.61 to 0.80 - substantial concordance; 0.81 to 1.00 - almost perfect concordance. Histopathology was defined as the gold standard for diagnosis. The diagnostic performances of the CEUS and CECT LI-RADS were evaluated using sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). The paired chi-square test was used to compare the sensitivity and specificity of the LI-RADS categories between the two imaging methods. The chi-square test was used to compare the PPV and NPV of the LI-RADS categories between the two imaging methods. Propensity score matching was further used to assess the diagnostic performance of CEUS and CECT LI-RADS in identifying benign and malignant hepatic lesion in order to reduce the effect of selection bias. The matched variables were age, gender, and tumor size. Differences were considered statistically significant at Table The comparison of the category-based results of CEUS and CECT LI-RADS is summarized in Table For CEUS LI-RADS, 7.3% 16/220) of the HCCs were categorized as either LR-3 or LR-4, 51.4% (113/220) as LR-5, and 41.3% (91/220) as LR-M. Three ICC and one metastasis were also classified as LR-5. For CECT LI-RADS, 19.5% (43/220) of the HCCs were categorized as either LR-3 or LR-4, 76.4% (168/220) as LR-5, and 4.1% (9/220) as LR-M. Four ICC, two CHC, and one metastasis were also categorized as LR-5. Twenty-two HCCs were classified as either LR-3 or LR-4 by CECT and LR-5 by CEUS; the average nodule size of these HCCs was 2.8 cm. Nine HCCs were classified as either LR-3 or LR-4 by CEUS and LR-5 by CECT; the average nodule size of these HCCs was 2.3 cm. Examples of the LI-RADS classification of FLLs in CEUS and CECT are shown in Figs. 20 of thep < 0.001) and 0.80 , respectively. The inter-modality consistency for CEUS LI-RADS and CECT LI-RADS was 0.31 . Table The inter-observer consistency for CEUS LI-RADS and CECT LI-RADS were 0.74 , 23.4%  vs. 95.5% , 70.3% . The diagnostic performances of CEUS and CECT LI-RADS for HCC and non-HCC malignancies in ≤ 5 cm FLLs are also shown in Supplemental Table 1.The diagnostic performances of the CEUS and CECT LI-RADS for hepatic malignancies are summarized in Table In this study, we compared the concordance and diagnostic performance of CEUS LI-RADS and CECT LI-RADS by analyzing 277 untreated FLLs in patients at risk for HCC. Our results indicated that both CEUS and CECT LR-5 perform well when it comes to diagnosing HCC. However, the fair concordance between the CEUS and CECT LI-RADS categories suggests that the same LI-RADS classifications cannot be assumed to fully equivalent; further analysis of their differences is needed. A meta-analysis of 11 studies that consisted of 5535 FLLs with 3983 HCCs showed that the specificity and PPV of CEUS LR-5 for diagnosing HCC were 92% and 97%, respectively . Anotherp < 0.001), and was primarily concentrated in small hepatic nodules. We found that arterial phase hyperenhancement and late and mild washout, all of which are important imaging features assigned to LR-5, were more frequently observed with CEUS than with CECT, suggesting that CEUS is more sensitive to these factors in FLLs than CECT is. CECT only scans at fixed time points, which could lead to potentially missing some key imaging features, while CEUS displays the early arterial enhancement features of FLLs in real time, essentially eliminating the possibility of misdiagnosis due to the time interval between CT scans in the arterial phase [In analyzing the category concordance of the two modalities, our results showed a fair concordance between CEUS LI-RADS and CECT LI-RADS; this is in line with previous findings , 7. In oal phase . In addial phase . This suIdentifying either the benign or malignant nature of FLLs is one of the important jobs of imaging. Although CEUS has significantly improved the diagnostic performance of conventional ultrasound in this regard, CT and MRI still dominate the field because CEUS has not been developed for a long time. This study showed that CEUS LI-RADS was more sensitive than CECT LI-RADS when CEUS and CECT LR-5 + LR-M were used as criteria for the diagnosis of hepatic malignancies, though differences in specificity, PPV, and NPV were not statistically significant. After further balancing the number of benign and malignant hepatic nodules, the assessment of diagnostic performance was performed again and similar results were obtained. This suggests that CEUS LI-RADS may be superior to CECT LI-RADS in terms of identifying the manifestations of hepatic malignancies. However, in distinguishing HCC from non-HCC malignancies, our study found that the diagnostic performance of CEUS LI-RADS was weaker than that of CECT LI-RADS. The specificity and PPV of CEUS LR-M for non-HCC malignancies was only 59.7% and 23.4%, as compared to 95.5% and 70.3% for CECT LR-M; both results were statistically significant. CEUS LR-M, at minimum, requires one of the subsequent three contrast features: rim arterial phase hyperenhancement, early washout (< 60 s), and marked washout < 120 s). We believe that it is primarily HCC  that is overrepresented in LR-M. Upon further analysis, we found that 82.4% (75/91) of HCCs were categorized as CEUS LR-M because of early washout alone; this is in line with previous studies , 18. Li  s. We beThis study had some limitations. First, our study was retrospective, which can lead to selection bias. Future prospective and multicenter studies are therefore necessary to validate these results. Second, this study used pathology as the gold standard, resulting in the inclusion of fewer benign hepatic lesions. In addition, if the diagnosis of one contrast-enhanced examination tends to be benign, it is generally unlikely that another contrast-enhanced examination will be performed at the same time, which will further reduce the number of benign hepatic lesions. Third, due to the operator-dependent nature of ultrasound, the CEUS LI-RADS v2017 makes it clear that it is currently only applicable to liver lesions that can be visualized by conventional ultrasound. The exclusion of liver lesions not visible on ultrasound or CT in this study will inevitably have some impact on the results, but we believe that the small number of patients excluded because of the invisibility of one of the modalities  will not affect the overall results.In conclusion, both CEUS LI-RADS and CECT LI-RADS showed strong diagnostic performances for HCC and were effective diagnostic tools for detecting HCC. In addition, the inter-modality consistency between the CEUS LI-RADS and CECT LI-RADS categories was fair. For other LI-RADS categories, such as CECT LR-3, LR-4, or CEUS LR-M, the selection of appropriate imaging examinations will help improve the diagnosis of HCC and non-HCC malignancies.Below is the link to the electronic supplementary material.Supplementary Material 1


Nowadays; intravitreal anti-vascular endothelial growth factor (VEGF) drugs are considered the first-line therapeutic strategy for treating macular exudative diseases; including wet age-related macular degeneration (w-AMD) and diabetic macular edema (DME). Despite the important clinical achievements obtained by anti-VEGF drugs in the management of w-AMD and DME; some limits still remain; including high treatment burden; the presence of unsatisfactory results in a certain percentage of patients and long-term visual acuity decline due to complications such as macular atrophy and fibrosis. Targeting the angiopoietin/Tie (Ang/Tie) pathway beyond the VEGF pathway may be a possible therapeutic strategy; which may has the potential to solve some of the previous mentioned challenges. Faricimab is a new; bispecific antibody targeting both VEGF-A and the Ang-Tie/pathway. It was approved by FDA and; more recently; by EMA for treating w-AMD and DME. Results from phase III trials TENAYA and LUCERNE (w-AMD) and RHINE and YOSEMITE (DME) have shown the potential of faricimab to maintain clinical efficacy with more prolonged treatment regimens compared to aflibercept (12 or 16 weeks) with a a good safety profile. Currently, wet-aged related macular degeneration (w-AMD) and diabetic retinopathy (DR) with diabetic macular edema (DME) are among the most frequent causes of visual impairment in the adult population in the Western countries ,2. In faThe current mainstay for the treatment of w-AMD and DME is represented by intravitreal drugs targeting the vascular endothelial growth factor (VEGF), which plays a crucial role in the pathogenesis of these exudative macular disorders ; howeverThe angiopoietin (Ang) yrosine kinase endothelial receptors (Tie) pathway has shown encouraging results for the management of w-AMD and DME .In adults, the Ang/Tie pathway is involved in the regulation of vascular homeostasis, the modulation of vascular permeability and in neoangiogenic and proinflammatory processes . In detaUnder physiological conditions, Ang-1 is constitutively expressed at higher concentrations than Ang-2 ; howeverInterestingly, preclinical studies highlighted that the simultaneous dual inhibition of Ang-2 and VEGF-A was superior to either anti–VEGF-A or anti–Ang-2 activity alone .Thus, the novel bispecific antibody, faricimab  targeting both VEGF-A and Ang-2, has been recently developed by Roche/Genentech for the treatment of w-AMD and DME . After mIn this review, we will discuss the pharmacological, safety and clinical efficacy profile of faricimab, and analyze its possible beneficial role in patients with w-AMD and DME.http://www.cliniclatrials.gov/ (accessed on 14 April 2023).A literature search was performed in order to find all the published studies dealing with pharmacodynamics, clinical efficacy, and safety of faricimab for treating w-AMD and DME from inception until February 2023. These following electronic databases were adopted: Medline, PubMed, Science Citation Index via Web of Science, and the Cochrane Library. The following search terms were used: ‘faricimab and ‘RG-7716’ alone or in combination with ‘age-related macular degeneration’, ‘diabetic macular edema’ ‘efficacy’, ‘safety’, ‘toxicity’. Current clinical trials on the efficacy and tolerability of faricimab were considered, by examining research registers such as 1/2) and to prevent the recruitment of immune cells. Moreover, the viscosity of the drug was reduced by introducing a Triple A mutation. The advantages of bispecific agents include the recruitment of effector cells, the reduction of systemic toxicities and modulation of internalization properties, and the presence of synergistic effects when cell-surface receptors are targeted  vs. aflibercept n = 128 [38.1%]) and LUCERNE  in the faricimab group and 1.8% (n = 6) in the aflibercept group in TENAYA and 2.1% (n = 7) in both groups in LUCERNE. In the large, multicenter, phase III TENAYA and LUCERNE trials, faricimab was well-tolerated. The overall percentage of patients discontinuing the therapy was low, with only 0.9% of the patients  and aflibercept  treated with faricimab had a visual loss greater than 30 letters; and only the 1 case with uveitis did not resolve completely at the time of primary analysis  and RHINE n = 113 [36%] in the bimonthly aflibercept group). Most of these ocular AEs were mild or moderate. Furthermore, the incidence of intraocular inflammation was low across both trials  versus bimonthly aflibercept n = 3 [1.0%], n = 1 [0.3%]).In the multicenter, phase III YOSEMITE and RHINE clinical trials, faricimab was proven to have an overall safe profile and it was well tolerated. The reported incidence of ocular AEs was comparable among all the treatment arms  .The ongoing, open-label, phase III trial Rhone-X is currently including DME patients previously included in YOSEMITE and RHINE, in order to analyze faricimab long-term safety and tolerability .In the multicenter, open-label, phase I clinical trial, 24 patients with w-AMD refractory to 3 or more anti-VEGF injections in the previous 6 months and a best-corrected visual acuity (BCVA) of 20/40 to 20/400  were injected with faricimab administered at single and multiple doses.The primary aim of the study was to assess the safety and tolerability of both single and multiple faricimab intravitreal doses. BCVA and central retinal thickness (CRT) changes were secondary outcomes. Faricimab was administered in a single dose during the single-dose phase at doses of 0.5 mg, 1.5 mg, 3 mg, and 6 mg, respectively. Six individuals were included in the multiple-dose phase and were given faricimab 3 mg and 6 mg.Patients received a single intravitreal administration of faricimab in the single dose phase only at day 1 and several intravitreal administrations scheduled on days 1, 28, and 56 in multiple-dose components.n = 1), with 91% of patients (n = 10/11) experiencing a visual gain of three letters or more. A good BCVA outcome was still observed at day 56, with a median change of +6 letters from baseline (n = 10/11). 28 days after the third injection, the 3-mg multiple-dose group had a median BCVA change from baseline of 0.5 letters , with 4 of the 6 patients maintaining baseline BCVA within 3 letters and 1 patient improving by 8 letters from baseline. The average BCVA deviation from baseline in the 6-mg multiple-doses group was +7.5 letters  was described 28 days after the third administration, with 5 of 6 patients experiencing at least a +5-letter improvement from baseline.On day 28, the average BCVA gain from baseline in the single-dose group was reported to be +7 letters  and −117 μm  in the same groups, respectively  in LUCERNE, +6.6 letters [5.3 to 7.8] were recorded in the faricimab group and +6.6 letters [5.3 to 7.8] in the aflibercept group.The mean modifications in CRT from baseline were −136.8 μm (95% CI −142.6 to −131.0) in the faricimab group and −129.4 μm (−135.2 to −123.5) in the aflibercept group in TENAYA (treatment difference −7.4 μm [−15.7 to 0.8]), and −137.1 μm (−143.1 to −131.2) in the faricimab group and −130.8 μm (−136.8 to −124.8) in the aflibercept group in LUCERNE (treatment difference −6.4 μm [−14.8 to 2.1]).In conclusion, visual benefits with faricimab administered with a max Q16W regimen in patients with w-AMD were comparable and non-inferior with those obtained with bimonthly aflibercept, which indicates the potential to extend the treatment interval without losing efficacy .n = 1532 in YOSEMITE and n = 1715 in RHINE) with visual impairment due to center-involving DME. The following treatment regimens were given to study participants by randomization (1:1:1): faricimab 6.0 mg every 8 weeks, faricimab 6.0 mg per PTI, or aflibercept 2.0 mg every 8 weeks. Based on disease activity, the PTI group had a variable treatment schedule (every 4 weeks (Q4W) up to Q16W). The mean change in BCVA at 1 year, averaged throughout weeks 48, 52, and 56, served as the main endpoint. The primary aim of both trials was to examine faricimab non-inferiority to aflibercept.YOSEMITE (NCT03622580) and RHINE (NCT03622593) were 2 multicenter, randomized, double-masked, phase III clinical studies, which enrolled 3247 patients  and +0.7 letters (97.52% CI 1.1 to 2.5)] and +1.5 letters (97.52% CI 0.1 to 3.2) and +0.5 letters (97.52% CI 1.1 to 2.1) in RHINE [treatment differences vs. aflibercept] and +11.8 letters, +10.8 letters, and +10.3 letters in the bimonthly faricimab, faricimab PTI and bimonthly aflibercept groups, respectively. The reductions in CRT were significantly greater in patients treated with faricimab than those treated with aflibercept after 12 months. In YOSEMITE, average CRT variations from baseline were −206.6 μm (95.04% CI −214·7 to −198.4) in the bimonthly faricimab, −196·5 μm (−204·7 to −188·4) in the faricimab PTI and −170.3 μm (−178.5 to −162·2) in the aflibercept groups, respectively. Similarly in RHINE, average CRT decreased from baseline by −195·8 μm (−204·1 to −187·5) in the bimonthly faricimab, −187·6 μm (−195·8 to −179·5) in the PTI faricimab versus −170·1 μm (−178·3 to −161·8) in the aflibercept groups, respectively. After 52 weeks, in both YOSEMITE and RHINE, 52% of patients randomized to the faricimab PTI group remained in a Q16W interval and 21% of them in a 12-week (Q12W) regimen. Thus, faricimab showed strong durability in YOSEMITE and RHINE, considering that more than 70% of patients in the PTI groups maintained in a ≥Q12W dosing regimen at 1 year.In conclusion, faricimab showed consistent visual and superior anatomical outcomes in patients with DME even in an extended the treatment regimen up to Q16W .Currently, the ongoing, multicenter, randomized, phase III BALATON (NCT04740905) and COMINO (NCT04740931) trials are investigating the clinical efficacy of faricimab in comparison with aflibercept and sham for the management of branch retinal vein occlusion (BRVO) and central retinal vein occlusion (CRVO), respectively. Preliminary results showed that monthly faricimab was non-inferior to monthly aflibercept in terms of visual function up to 24 weeks ,42.In addition, the ongoing, multicenter, single-arm, phase IV ELEVATUM trial is studying the clinical response to faricimab in treatment-naïve patients with DME, which were underrepresented in the previous phase II and III trials .The complete list of the clinical trials evaluating the clinical efficacy of faricimab for treating w-AMD, DME and BRVO/CRVO is summarized below .Although anti-VEGF drugs still represent the mainstay for the treatment of exudative retinal disorders, including w-AMD and DME, other new therapeutic strategies are being examined with the aim to reduce the treatment burden and improve the efficacy and durability of intravitreal injections ,10,45,46®, Montreal, QC, Canada), ranibizumab , aflibercept  and only for w-AMD brolucizumab ; moreover, another anti-VEGF, bevacizumab, is largely adopted in clinical practice as an ‘off-label’ treatment option [Currently, there are 4 intravitreal anti-VEGF agents approved by the USA FDA and EMA for treating w-AMD and DME: pegaptanib sodium , a bispecific antibody with a molecular structure that binds to both VEGF-A and Ang-2 with high affinity, was recently licensed by the FDA and EMA . AccordiThe following large, multicenter, phase III clinical trials RHINE and YOSEMITE for DME and LUCERNE and TENAYA for w-AMD have provided evidence in a clinical setting that faricimab shows prolonged efficacy up to a Q16W treatment regimen, without being inferior to aflibercept in terms of visual and anatomical outcomes ,38.In terms of visual function, a fast and sustained improvement in BCVA was achieved with a faricimab dosing of either Q8W or PTI after 52 weeks, which was comparable to the visual function improvement in the Q8W aflibercept group. In the PTI groups, non-inferiority was obtained with fewer interval-determining visits as opposed to aflibercept .In LUCERNE and TENAYA rapid BCVA gains were sustained up to week 48 and a comparable percentage of patients with w-AMD improved of 15 letters or more in both faricimab and aflibercept groups. In TENAYA 79.9% of the patients completed the 48-week follow-up period with a 12-week or 16-week regimen and similarly did 77.8% of the patients in LUCERNE. This high percentage reflect the capability of faricimab to prolong the treatment regimen without losing efficacy in a big part of the patients in comparison with aflibercept (8-week regimen) .In RHINE and YOSEMITE a higher percentage of patients with DME showed absence of fluid in comparison with the aflibercept-treated subjects up to week 56 . In thisResults from these large, multicenter trials suggest the potential of faricimab to achieve stable and durable results and the possibility to tailor a personalized treatment regimen in relation to individual needs, with important benefits in the reduction of the treatment burden and costs for both healthcare providers and patients. Further real-life clinical studies on faricimab should also assess the possible presence of gender, ethnic or age differences in the treated population; in fact, previous studies on anti-VEGF agents have reported the presence of gender and racial differences in the prevalence and disease severity in patients affected by DME and w-AMD .Furthermore, it has been reported the negative effect of the dynamic fluctuations in CRT in patients with DME and w-AMD treated with anti-VEGF agents; in fact these dynamic changes are associated with poorer visual outcomes ,52. FariOverall, faricimab has so far shown a good safety and tolerability profile, with an incidence of ocular (including intraocular inflammation) and systemic AEs comparable to that of aflibercept . Also, nA potential limitation of phase III clinical trials is that they only compared faricimab with aflibercept; in the near future, further studies should provide evidence on the direct comparison between faricimab and the other approved anti-VEGFs. Moreover, results of the comparative analysis of faricimab show similar or non-inferior responses to aflibercept rather than showing superior therapeutic responses. Further real-life clinical studies should provide more evidence on the long-term anatomical and visual outcomes of faricimab in patients with w-AMD and DME.This is in contrast with the recent FDA and EMA- approved brolucizumab , a single-chain antibody fragment targeting VEGF-A. Although, brolucizumab has shown a great clinical efficacy in restoring BCVA and maintaining the absence of intraretinal and/or subretinal fluid in pivotal trials (HAWK and HARRIER) and in real-life studies, some safety concerns have been raised regarding the higher risk of intraocular inflammation and, in some cases, retinal vasculitis and retinal vascular occlusions ,49. In fAnother treatment modality targeting the Ang/Tie has been investigated for treating w-AMD. This consists of intravitreal nesvacumab plus aflibercept combination therapy a fixed dose providing specific doses of the anti-Ang2 antibody with a known effective dose of aflibercept at 2 mg. In the phase II RUBY (NCT02712008) trial, the primary endpoint was the mean change in BCVA after 12 weeks. The primary endpoint was to demonstrate the superiority of nesvacumab combination therapy than aflibercept alone. They reported better anatomical outcomes, but not visual benefit (which was the primary outcome) with combination therapy in comparison with aflibercept. Thus, since nesvacumab therapy did not show to be superior to aflibercept therapy, no phase III trials were continued .1/2, which, according to the phase II GLEAM and GLIMMER clinical trials, may lead to an extension of the treatment regimen up to 16–20 weeks; however, recent partial results have shown that KSI-301 did not meet the primary endpoint of non-inferiority to aflibercept for treating w-AMD; however, further data are needed to provide more evidence on KSI-301 clinical efficacy [Currently, together with faricimab, other novel drugs are being investigated for the management of w-AMD and DME ,59. KSI-efficacy .Furthermore, the ranibizumab port delivery system (RPDS), which is a surgically-implanted device allowing the continuous delivery of ranibizumab into the vitreous for 6 months or more, has been approved for treating w-AMD and is under investigation in phase 3 trials for DME ; howeverDifferently, abicipar pegol, a small DARPin protein targeting VEGF-A, despite the satisfactory clinical results has not been approved due to the high incidence of intraocular inflammation .Currently, also gene therapy thorough the long-lasting VEGF blockage is attracting interest for the treatment of w-AMD; RGX-314, ADVM-022, and ADVM-032 are the most promising options in this regard . In thisAlso the TGF-beta is being examined in early-phase clinical trials in order to prevent the fibrotic stage of macular edema in patients with w-AMD and DME . ResultsOther early-phase studies are investigating the role of tyrosine kinase inhibitors, targeting VEGFR, PDGFR, fibroblast growth factor receptor (FGFR), and c-kit against CNVs. In this regard, a phase I study on oral X-82, targeting both VEGF and PDGF, showed in 35 patients with w-AMD that 60% of them did not need further anti-VEGF agents during the follow-up period. A side effect was the temporary elevation of liver enzymes . FurtherIn conclusion, the modulation of Ang/Tie pathway with faricimab has proven to be a potential first-line treatment option in the management of w-AMD and DME, allowing to decrease the treatment burden and extend the regimen up to 16 weeks.Further clinical trials are needed to better understand faricimab efficacy and safety in both treatment naïve-patients with DME and w-AMD and in those patients already treated with anti-VEGF agents, which were underrepresented in pivotal, phase III clinical trials (RHINE and YOSEMITE for DME and TENAYA and LUCERNE for w-AMD).


The Portsmouth-Physiological and Operative Severity Score for the enumeration of Mortality and Morbidity (P-POSSUM) is one of the scores that is used most frequently for determining the likelihood of mortality in patients undergoing emergency laparotomy. National Emergency Laparotomy Audit (NELA) presents a novel and validated score. Therefore, we aimed to compare the performance of the NELA and P-POSSUM mortality risk scores in predicting 30-day and 90-day mortality in patients undergoing emergency laparotomy.Between August 2020 and October 2022, this cohort study was undertaken at Menoufia University Hospital. We compared the P-POSSUM, preoperative NELA, and postoperative NELA scores in patients undergoing emergency laparotomy. All variables needed to calculate the used scores were collected. The outcomes included the death rates at 30 and 90 days. By calculating the area under the curve (AUC) for every mortality instrument, the discrimination of the various methods was evaluated and compared.Data from 670 patients were included. The observed risk of 30-day and 90-day mortality was 10.3% (69/670) and 13.13% (88/670), respectively. Concerning 30-day mortality, the AUC was 0.774 for the preoperative NELA score, 0.763 for the preoperative P-POSSUM score, and 0.780 for the postoperative NELA score. Regarding 90-day mortality, the AUCs for the preoperative NELA score, preoperative P-POSSUM score, and postoperative NELA score were 0.649 (0.581–0.717), 0.782 (0.737–0.828), and 0.663 (0.608–0.718), respectively. There was noticeable difference in the three models' capacity for discrimination, according to pairwise comparisons.The probability of 30-day and 90-day death across the entire population was underestimated by the NELA and P-POSSUM scores. There was discernible difference in predictive performance between the two scores. Emergency laparotomy results in approximately 10 times more fatalities than major elective gastrointestinal surgery, which is a frequent urgent surgical procedure , 3. BecaThis prospective cohort study was conducted at the general surgery department, Menoufia University Hospital, Egypt. All adult patients aged over 18 who underwent emergency laparotomy between 1st August 2020 and 1st October 2022, were deemed eligible for this study. Patients under the age of 18, patients underwent laparotomies or laparoscopies on an elective basis and those who did not have all the required investigations to calculate the NELA or P-POSSUM scores were excluded. Patient data such as age, sex, comorbidities, clinical examination, laboratory investigations especially , ECG, chest X-ray, American Society of Anesthesiology (ASA) score, Glasgow coma scale, clinical urgency, surgical indications and operative parameters needed to determine the NELA and P-POSSUM scores, as well as the 30- and 90-day mortality, were gathered and analyzed , 11. ProThe sample size was calculated to include all patients who underwent emergent laparotomy, fulfilling all inclusion and exclusion criteria, and were collected from Menoufia University hospitals over two years. The least sample size calculated using statistics and sample size pro was 416 participants. The power of the study is 80% and the confidence interval is 95% .The statistical analysis was performed with SPSS Statistics version 25 by IBM Corp. . The categorical variables were summarized using absolute and relative frequencies and compared using the chi-square test. The continuous variables were summarized using the median and compared using the Monte Carlo test and Mann–Whitney U test. For every predicted model, we determined the observed deaths to expected deaths ratio (O/E ratio) by dividing the actual mortality rate (observed) by the calculated mortality score (expected). The O/E ratio would help assess whether the risk-prediction tool overestimates or underestimates the risk of postoperative mortality. Analyses using the receiver operating characteristic (ROC) curve were carried out to measure and contrast the mortality tools' discriminatory abilities by calculating the AUC for every mortality tool. Diagnostic Odds Ratio (DOR) and Bootstrap were used to estimate the uncertainty in AUC and other performance metrics. At the 5% level of significance, the results were found to be substantial.A total of 724 patients underwent emergency laparotomy at our hospitals between August 2020 and October 2022. 54 patients of them were excluded because they did not have all the required investigations to calculate the NELA or P-POSSUM scores. Therefore, only 670 patients were included in our current study. Patients` age was from 18 to 91, and 45.7% (306/670) of them were male. The classification of the ASA score is shown in Table For 90-day mortality, the estimated observed risk was 13.13% (88/670) while for 30-day mortality it was 10.3% (69/670). The predicted mortality hazard was determined using the preoperative NELA score, preoperative P-POSSUM score, and postoperative NELA score. The results were 3.2% (0.01–84.9), 3.7% (0.01–96.7), and 4.8% (0.01–98.7), respectively. The O/E ratio for 30-day mortality was measured as 3.25 for preoperative NELA, 2.81 for preoperative P-POSSUM, and 2.17 for postoperative NELA. The O/E ratio for 90-day mortality was determined to be 4.43 for preoperative NELA, 3.84 for preoperative P-POSSUM, and 2.96 for postoperative NELA. The aforementioned results demonstrated that all models . As regard to the Diagnostic Odds Ratio by binary logistic regression for mortality scores, the postoperative NELA at 30 days was risky which each unit increase of postoperative NELA score, the risk of mortality occurred by 2.328 times  = 0.719–0.829, p 0.001), the preoperative P-POSSUM score was 0.763  and the postoperative NELA score was 0.780   and the postoperative NELA score was 0.663 , according to ROC curve analysis  but there was statistical significant difference between preoperative NELA score and postoperative NELA score, and preoperative P-POSSUM score and postoperative NELA score (p = 0.001) , the preoperative NELA score was 0.649  which means that the 47.8% of mortality belong to mortality scores parameters at 90 days. The probability of mortality at 90 days due to mortality scores parameters was higher than at 30 days  , so the estimation of mortality at 90 days was more accurate than 30 days (Figs. Risk assessment plays a significant role in identifying surgical patients at high risk and in setting treatment standards that are appropriate for those risks. One way of identifying high-risk patients is mortality estimates based on risk models such as the NELA and P-POSSUM scores, which may be utilized to determine patients at increased risk and induce their enrollment in hospitals with superior levels of care. We underestimated the risks of 30-day mortality and 90-day mortality in the current study by comparing the effectiveness of the NELA mortality hazard score and the P-POSSUM model in cases experiencing emergency laparotomy. The ability of before-surgery NELA, before-surgery P-POSSUM, and after-surgery NELA scores to predict death after 30 days and death after 90 days in the 670 participants differed statistically significantly, according to pairwise comparisons. Numerous studies , 14 haveUsing comparison data from the current study and prior research, it can be concluded that the P-POSSUM model and the NELA model are equally good at forecasting outcomes. Lai et al. recommended that the NELA score be used instead of the P-POSSUM model since death rates were inflated and associated with the NELA score were lower than those associated with the PPOSSUM model . Both ouThere are some limitations in our study such as it might not be the best representative of the entire population because it was a single-center study. Moreover, we excluded 54 patients because they did not have all the required investigations to calculate the NELA or P-POSSUM scores; this should be taken into account when interpreting our findings. On the other hand, the sample size was relatively large enough to determine the accuracy of the performance of the NELA and P-PPOSSUM mortality models. Also, the consideration of 90-day mortality in addition to 30-day mortality and evaluation of the performance of postoperative NELA score beside the performance of preoperative NELA and PPOSSUM scores were the strengths of this study.In the current study, we demonstrated that there were appreciable variances between the 30-day and 90-day mortality predictions given by preoperative NELA, preoperative P-POSSUM, and postoperative NELA scores. The likelihood of death between 30 and 90 days was underestimated by all models. Patients undergoing emergency laparotomy have a high mortality rate, thus future research should focus on developing and verifying scoring systems that account for all of these parameters.


The Cu-filling process in through-silicon via (TSV-Cu) is a key technology for chip stacking and three-dimensional vertical packaging. During this process, defects resulting from chemical–mechanical planarization (CMP) and annealing severely affect the reliability of the chips. Traditional methods of defect characterization are destructive and cumbersome. In this study, a new defect inspection method was developed using Mueller matrix spectroscopic ellipsometry. TSV-Cu with a 3-μm-diameter and 8-μm-deep Cu filling showed three typical types of characteristics: overdishing (defect-OD), protrusion (defect-P), and defect-free. The process dimension for each defect was 13 nm. First, the three typical defects caused by CMP and annealing were investigated. With single-channel deep learning and a Mueller matrix element (MME), the TSV-Cu defect types could be distinguished with an accuracy rate of 99.94%. Next, seven effective MMEs were used as independent channels in the artificial neural network to quantify the height variation in the Cu filling in the z-direction. The accuracy rate was 98.92% after training, and the recognition accuracy reached 1 nm. The proposed approach rapidly and nondestructively evaluates the annealing bonding performance of CMP processes, which can improve the reliability of high-density integration. TSV technology involves several processes, including etching holes in Si chips, depositing insulating/blocking/seeding layers, filling blind holes with Cu conductors, removing the backside Si and Cu overlay films via chemical–mechanical planarization (CMP) to expose Cu microcartridges, and ball bonding9. Several researchers have investigated the TSV manufacturing process. The microcharacterization and nanocharacterization of various processes have also received considerable attention in improving the reliability of 3D ICs.In recent years, three-dimensional (3D) integrated circuit (IC) technology with through-silicon via (TSV) has attracted significant attention because of its versatility, small size, and high performance. 3D IC is a technology that reduces the overall wire length and delay by vertically stacking multiple chips through high-density chip-to-chip interconnects12. Recently, studies on the Cu-filling process in TSV (TSV-Cu) protrusion have been conducted15. In these processes, detailed physical analyses are required because of the considerable impact of TSV-Cu protrusion on the reliability of the final 3D stacking. Many studies have been conducted to investigate annealing Cu protrusion and CMP dishing23. Cu protrusion and excessive dishing reduce the reliability of 3D stacking. In TSV manufacturing, it is crucial to control the CMP speed, annealing time, and temperature. Insufficient or excessive wafer polishing can lead to leaks and shorts, making the chips defective. Residual stress, interface delamination, and cracking occur when the annealing time or temperature is insufficient or excessive26. However, real-time nondestructive characterization methods for Cu protrusions and dishing remain limited.Significant issues in TSV technology include residual stress, extrusion, cracking, delamination, and Cu leaks, leading to severe reliability problems, such as warping of chips or wafers, interface delamination, cracking, and weak internal wire contact. These problems mainly result from the significant difference in the coefficient of thermal expansion between the surrounding silicon substrate and the TSV filler metal9. The period and critical size of the TSV-Cu structure prevail on the microscale for both vertical and horizontal dimensions. However, during CMP and annealing processes, the accuracy of the z-direction of Cu must be controlled at the nanoscale24. Cross-scale defect characterization in the z-direction of TSV-Cu structures is therefore a significant challenge.Nondestructive characterization of TSV-Cu in a production line has not been extensively investigated. When many defects appear during the TSV process, the detection time obtained using conventional methods is unsatisfactoryIn this study, an ellipsometry measurement method was developed for the characterization of TSV-Cu. The rigorous coupled-wave analysis (RCWA) algorithm was employed to calculate the reflection electric field of TSV-Cu with different dimensions of Cu filling in the z-direction at different wavelengths. The corresponding Mueller matrices were calculated by modulating the incident and reflective electric fields, as described in the “Methods” section. Deep learning with Mueller matrix datasets (see the “Methods” section for details) was used to distinguish the different TSV-Cu defects. The effects of the annealing temperature and time on the Cu grain size were experimentally studied, and the defect size of TSV-Cu was quantified using a multichannel deep-learning method. A single Mueller matrix element (MME) was replaced with multiple MMEs as a dataset to improve the stability and accuracy of the quantitative TSV-Cu size.2 as an insulating layer, a silicon substrate, and a Cu-filling layer with a diameter of 3 μm and depth of 8 μm. The Cu-filling period was 6 μm, the incidence angle was fixed at 45°27, and the wavelength range was 400–1000 nm. The scanning electron microscopy images of the TSV-Cu cross-section are shown in Fig. Hu) changes due to the significant mismatch of the coefficient of thermal expansion (CTE) between the Cu-filling and silicon substrate. Due to the higher CTE of Cu, the expansion of Cu is greater than that of silicon. At this temperature, there is almost no change in the microstructure of Cu-filling. The recrystallization of Cu-filling occurs at approximately 200–250 °C, at which point the Cu grain is refined. Once the annealing temperature exceeds 250 °C, the grain grows significantly as driven by the total free energy3. Hu is positively correlated with the grain size14. Therefore, Hu increases subsequentially. During the CMP process, the removal of the barrier layer and insulating layer is slower than that of Cu filling. Through this process, Hu decreases subsequentially. When Hu is excessively large or small, there is a risk of cracks and voids, respectively. Under the effect of poor bonding quality, the electric and thermal resistance of TSV-Cu increase, and the surface bonding strength decreases. Therefore, a reasonable Hu process window (−16 to −4 nm) is proposed in this work to achieve void-free and reliable interconnection13. A schematic of the defect types is shown in Fig. Hu exceeded 0 nm or was lower than 4 nm in the z-direction, it was considered a protrusion defect (defect-P). Hu ranging between 4 and 16 nm was considered free from defects (defect-free). Hu exceeding 16 nm indicated an overdishing defect (defect-OD). Defect-P and defect-OD caused extrusion cracking and incomplete contact during annealing bonding, respectively. The actual process window (4–16 nm) was expected to have a higher upper limit because no increase in the dielectric bond strength was assumed.Figure m11. The ordinate of each photograph represents Hu, and the abscissa represents the wavelength. The off-diagonal MMEs were nearly zero because the structure of TSV-Cu was isotropic28. The sensitivity of the MME, m12, with a change in the structure, increased at 0.7–1.0 μm wavelengths. The sensitivities of m21 and m22 with structural change increased at 0.85–1.05 μm wavelengths. The sensitivities of m33 and m44 with structural change increased at wavelengths of 0.80–1.05 μm. In addition, the sensitivities of m34 and m43 with structural change increased at 0.80–0.95 μm wavelengths. We selected seven effective MMEs  as the sample set for defect classification, ignoring the off-diagonal MMEs. The effective MMEs corresponding to 13 structures with Hu ranging from −4 to −16 nm , −17 to −29 nm , and −3 to 9 nm  and the 300 noise dataset of each structure were adopted as the artificial neural network (ANN) datasets representing the defect-free, defect-OD, and defect-P topologies, respectively.Figure m12 was faster than those of the other elements, with an increase in training time. The Mueller matrices can be decomposed using the Lu–Shipman polar decomposition method29:D is the dichroic scalar of the Mueller matrices after polar decomposition, and Hu values showed obvious changes. In defect classification training using m12, the verification accuracy at the seventh epoch was 99.80%, and the corresponding cross-entropy loss was 0.00043. Hence, only a single MME was required as a sample set to complete the defect classification using deep learning. t-distributed stochastic neighbor embedding (t-SNE) was employed to analyze the defect types, as shown in Fig. 3c30. Different scatter colors represent different defect types, and each point represents the simulation data with 10% noise randomly generated by m12 . The convergence rate of the multichannel training cross-entropy loss and validation accuracy was faster than that of single-channel training. Figure Top-Bottom) of the Cu-filling. The diameter of the Cu-filling is 3 μm, the aspect ratio is 1, and the ΔTop-Bottom values are 2 μm, 1.6 μm, 1.2 μm, 0.8 μm, 0.4 μm, 0.2 μm, and 0 μm . The relationship between the reflected field and wavelength of the structural, optical model representing the periodic grating was calculated. Ss, can be expressed as follows:δ is the phase difference between Ex and Ey. The light reflected by the sample is remodulated as Sout:MA is the Mueller matrix of the analyzer, m1132.The reflected electric fields, 34. Each CNN layer was connected to a max-pooling layer with batch normalization. The number of filters and kernel size of each channel in each CNN layer are listed in Table Hu at different wavelengths were introduced into the ANN input layer as parallel channels, and the kernel size of each channel was seven. Each defect type was assigned to a neuron in the ANN output layer. During the training of the ANN, a Mueller matrix was introduced into the ANN input layer, and different defect types were introduced into the output layer. Hundreds or tens of sample sets for each group of structures were not produced because of the cost and time in the actual TSV-Cu production process. During the process of sample measurement, there are two random errors, including the sample manufacturing error and measurement error36. The manufacturing error of surface displacement fluctuation of 0.1 nm is inevitable in the TSV-Cu process23. It is difficult to obtain specific measurement error because the measurement error depends on the instrument and environment. In practice, the spectral change caused by measurement error is usually smaller than that caused by manufacturing error. The spectrum of TSV-Cu surface fluctuation (Hu ± 0.1 nm) was analyzed, and all effective spectral change rates were less than 10%  was employed, matching a fully connected network (FCN) Fig. . The “le10% Fig. . It can